BRAFV600E-papillary thyroid carcinoma (V600E-PTC) is particularly aggressive, and as it deregulates iodine metabolism, its victims are unresponsive to radioiodine treatment, and suffer high rates of metastasis and recurrence, and low survival rates. BRAFV600E inhibitors such as vemurafenib (vemu), which at first showed clinical promise, are the only therapeutic option, but resistance to these drugs is widely reported. Our premise is that the epigenetic silencing of the first identified thyroid-specific long intergenic non-coding RNA (lincRNA), Xloc001313 (Xloc13) deregulates iodine metabolism, sustains tumor cell survival, and promotes progression and drug resistance. Xloc13 is active downstream of the coding gene for TPO, a key enzyme for iodine metabolism, and its expression is far lower in V600E-PTC than normal tissue. Our project is designed to determine the cellular mechanisms behind these effects, and use these findings to better target clinical trials of new treatments. Vemu treatment induces Xloc13 expression, indicating it is silenced by BRAFV600E, and its inactivation by CRISPR in normal thyroid cells reduces TPO levels and iodine uptake, while increasing TSP-1 levels (extracellular matrix protein) and cell proliferation. Xloc13 RNA enhancer maps to DNA-binding motifs on the TSP-1 promoter, leading to down-regulation of TSP-1, which inactivates TGF?1 to inhibit PTC cell and pericyte viability. We recently demonstrated that V600E-PTC samples are enriched in pericytes that co- express TSP-1 and TGF?1 to sustain V600E-PTC cell survival. Xloc13 silencing correlates with high pericyte density, linking its action to pericyte biology. We also identified regulatory motifs in the Xloc13 promoter for EZH2 and E2F1 as transcriptional repressors up-regulated in vemu-resistant V600E-PTC cells and ChIP- enriched at these DNA-binding motifs. Critically, combining inhibitors of BRAFV600E+EZH2+CDK4/6 durably rescued Xloc13, and reduced PTC cell and pericyte viability. Xloc13 down-regulation therefore plays a key role in V600E-PTC, and our central objective is to determine its mechanisms of regulation and their impact on tumor progression and resistance. We hypothesize that BRAFV600E synergizes with EZH2 and the CDK4/6 (upstream of E2F1) pathway to down-regulate Xloc13, which deregulates iodine metabolism, enhances tumor viability, and increases invasion signaling through pericyte action, which leads to secondary drug resistance and tumor progression. Using cutting-edge technologies (CRISPR, ChIP, ATAC, RAP) and new cancer mouse model, our specific Aims are: 1) To identify the mechanisms regulating Xloc13 in V600E-PTC and their impact on tumor aggressiveness; 2) To determine the role of Xloc13 in the regulation of TPO in V600E-PTC and its impact on iodine metabolism and PTC cell survival; 3) To assess the efficacy of anti-BRAFV600E-EZH2-CDK4/6 combined therapy on human V600E-PTC in vivo. Our studies will have high clinical significance by supporting new clinical trials of trimodal combined therapies for advanced V600E-PTC patients, and identifying new mechanisms in PTC and its microenvironment which could serve as drug development targets for this tumor.